Melphalan, (4-[bis(2-chloroethyl)amino]-L-phenylalanine), is a nitrogen mustard that is useful as a chemotherapeutic agent. Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, 8th edition, pages 1202-1208 (1990) classifies melphalan as an alkylating agent type of chemotherapeutic action and indicates a mechanism of action of cross-linking DNA. Sarosy, G., et al, Journal of Clinical Oncology, Vol. 6, No. 11 (November), pp. 1768-1782 (1988) indicates that melphalan effects cytotoxicity by forming either interstrand, intrastrand, or DNA-protein cross links.
The wide spectrum of melphalan's anti-neoplastic activity against tumors, in vivo, is reported in the literature. Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, 8th edition, pages 1202-1208 (1990) indicates that melphalan is currently used in the treatment of multiple myeloma, breast cancer and ovarian cancer. Sarosy, G., et al, Journal of Clinical Oncology, Vol. 6, No. 11 (November), pp. 1768-1782 (1988), a review article on intravenous melphalan usage, at page 1772 in Table 1 indicates that at lower doses intravenous melphalan demonstrated at least some activity against pancreatic cancer, colon carcinoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, and ovarian cancer; at page 1774 in Table 3 indicates that at higher dosages intravenous melphalan demonstrated at least some activity against breast cancer, non-small-cell lung cancer, small-cell lung cancer, colon cancer, melanoma, testicular cancer, ovarian cancer, soft tissue sarcoma, Ewing's sarcoma, synovial cell sarcoma, bone (giant cell) sarcoma, Wilms' sarcoma, Wilms' osteogenic sarcoma, rhabdomyosarcoma, multiple myeloma, neuroblastoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, acute nonlymphoblastic leukemia, chronic granulocytic leukemia and renal cancer and characterizes the response rate for melanoma and colon carcinoma as extraordinarily high; at page 1773 in Table 2 indicates that drug combinations including low dosages of intravenous melphalan demonstrated at least some activity against ovarian cancer, testicular cancer, non-small-cell lung cancer, melanoma and multiple myeloma; and at pages 1776-177 in Table 4 indicates that drug combinations including higher dosages of intravenous melphalan demonstrated at least some activity against neuroblastoma, melanoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkett's lymphoma, CML-blast crises, multiple myeloma, colonic cancer, breast cancer, sarcoma and gastric cancer. Barlogie, B., et al, Blood, Vol. 67, No. 5 (May), 1298-1301 (1986), indicates that large doses of melphalan demonstrated activity against advanced multiple myeloma. Horowitz, M. E., et al, Journal of Clinical Oncology, Vol. 6, No. 2 (February), 308-314 (1988), indicates that melphalan demonstrated partial responses in 10 of 13 having newly diagnosed, poor-risk rhabdomyosarcoma. Houghton, J. A., et al, Cancer Treatment Reports, Vol. 69, No. 1, 91-96 (1/85), indicates that melphalan demonstrated complete regressions in 6 of 7 lines of childhood rhabdomyosarcomas. Leff, R. S., et al, Journal of Clinical Oncology, Vol. 4, No. 11 (November), pp. 1586-1591 (1986), indicates that high-dose melphalan demonstrated complete responses in 15% of cases of metastatic colon cancer and partial responses in 30% of cases of metastatic colon cancer. Pritchard, J., et al, Br. J. Cancer, 45, 86-94 (1982), indicates that high dose melphalan demonstrated complete response in 6 of 11 of certain patients with advanced neuroblastoma.
With cells grown in culture, melphalan has been shown to be effective against brain tumors, including gliomas, and medulloblastomas. See Friedman, H. S., et al, Cancer Research 46, 2817-2838, 6/86 and Friedman, H. S., et al, Cancer Research 48, 4189-4195, 8/88 on the experimental chemotherapy of human medulloblastoma cell lines.
It has been discovered that starving followed by a protein-free diet reduces plasma levels of protein amino acids including large neutral amino acids and increases the blood-to-tumor periphery tissue transfer constant of melphalan both for subcutaneous tumors (representative of all tumors except for brain tumors) and also for brain tumors. See Friedman, H. S., et al, Proceedings of the American Association for Cancer Research, Volume 32, page 318, Abstract 1886, March, 1991 and Groothius, D. R., et al, Cancer Research 52, 5590-5596 (Oct. 15, 1992). This increase of blood-to-tumor tissue transfer constant might be expected to allow use of lesser dosages of melphalan (and concomitant reduced toxicity) in circumstances where melphalan is now considered useful and the extension of use of melphalan in circumstances now foreclosed by the blood brain barrier, i.e., as an antitumor agent against brain (intracranial) tumors. However, the accomplishment of this by means of starving and administration of a protein free diet affords at most limited improvement.
The finding that reduced plasma levels of large neutral amino acids were associated with increased blood-to-tumor melphalan transfer constants is consistent with previous work showing that melphalan is transported by the same transporter as the large neutral amino acids and that the presence of large neutral amino acids in plasma interferes with the transport of melphalan. Thus achieving reduction of plasma levels of large neutral amino acids by means different from or additional to restricted diet to the same or greater degree as is obtained with said restricted diet, should improve melphalan transport and result in a benefit if said different means doesn't concurrently provide deleterious effect.
Various enzymes are known for which large neutral amino acids are substrates and which would be useful for reducing plasma levels of these provided they have access to required cosubstrates. However, melphalan is also an amino acid and would likely be a substrate for the same enzymes. Furthermore, the various possible enzymes would be expected to differ in respect to the number of different large neutral amino acids that would be substrates and in their relative kinetic constants vis-a-vis their large neutral amino acid substrates and melphalan. Therefore, such enzymes might be expected, on the one hand, to potentiate the transport of melphalan into tumors by reducing plasma concentrations of large neutral amino acids, but on the other hand, would be expected to act in counterproductive fashion if still present upon melphalan administration by degrading melphalan to an extent which might be larger than the extent of increased melphalan transport from large neutral amino acid depletion. Furthermore, each particular enzyme might be expected to have a different effect on concentrations of plasma large neutral amino acids and degradation of melphalan.
What is necessary is selection of an enzyme which will reduce plasma large neutral amino acids to enhance melphalan transport but which would be relatively less active toward melphalan or which would or could be sufficiently inactivated, within the period of reduced plasma amino acid level, so as not to degrade the melphalan to an extent of negating the benefit obtained by enhanced melphalan transport. For any particular enzyme, there is no expectation of success of meeting these criteria.